Endophyte enhanced seedlings with increased pest tolerance

• US 8,455,395 B2
• Filed: 10/24/2007
• Issued: 06/04/2013
• Est. Priority Date: 10/24/2006
• Status: Active Grant

• Current Assignee: Irving Licensing Inc.
• Original Assignee: Irving Licensing Inc.
• Inventors: Miller, John David, Adams, Greg William
• Primary Examiner(s): Kruse, David H
• Application Number:
  US12/447,217
• Publication Number:
  US 20110173727A1
• Time in Patent Office: 2,050 Days
• Field of Search: None
• US Class Current: 504/117
• CPC Class Codes: A01H 17/00 Symbiotic or parasitic combinations including one or more new plants, e.g. mycorrhiza lichens A01H15/00
• CPC Class Codes: A01H 3/00 Processes for modifying phenotypes , e.g. symbiosis with bacteria A01H4/00 takes precedence
• CPC Class Codes: A01H 7/00 Gymnosperms, e.g. conifers
• CPC Class Codes: A01N 63/30 Microbial fungi; Substances produced thereby or obtained therefrom
• CPC Class Codes: C07K 16/14 against material from fungi, algea or lichens
• CPC Class Codes: C12N 1/145 Fungal isolates
• CPC Class Codes: C12R 2001/645 Fungi ; Processes using fungi
• CPC Class Codes: G01N 33/56961 Plant cells or fungi

Abstract

The invention provides methods for preparing a conifer seedling with increased tolerance to a pest. A conifer seedling is inoculated with an isolated endophyte when the conifer seedling is susceptible to colonization by the endophyte.

Claims

1. A method of preparing a conifer seedling with increased tolerance to an insect or fungal pest, comprising:

a) inoculating a nursery grown conifer seedling with an isolated toxigenic endophyte that can colonize the conifer seedling, during a susceptible time window, wherein the nursery conifer seedling is inoculated by spraying on the conifer seedling liquid particles of an inoculum composition comprising the isolated toxigenic conifer endophyte and water, so that at least a portion of the toxigenic endophytes and water contact the conifer seedling in an environment of wetness, he inoculating step occurring during the susceptible time window that is post germination during sustained elongation of the shoot apex, when the conifer seedling'"'"'s percentage of needles of intermediate differentiation is greater than the percentage of needles of complete differentiation in which the cuticle is fully formed; and

b) drawing the conifer seedling to obtain the conifer seedling with increased pest tolerance to the insect or fungal pest,

wherein the increase tolerance is relative to conifer seedling grown from an uninoculated conifer seedling; and

wherein the toxigenic endophyte is selected from the group of toxigenic endophytes deposited with Centraalbureau voor Schimmelcultures (CBS) international depository agency in the Netherlands, having the accession numbers consisting of CBS 120377, CBS 120378, CBS 120381, CBS 120379, CBS 120380, CBS 121946, CBS 121945, CBS 121944, CBS 121943 and CBS 121942.

2. The method of claim 1, wherein the toxigenic endophyte is toxigenic endophyte hyphal fragments and the inoculum composition comprises at least 1-25 toxigenic endophyte hyphal fragments/6 microliter.

3. The method of claim 1, wherein the spraying method is selected from the group of misting, ground spraying, bottle spraying, and boom spraying.

4. The method of claim 1 comprising repeating the inoculation step a), during the susceptible time window.

5. The method of claim 1, further comprising determining whether the endophyte has colonized the seedling by contacting a sample of the seedling with a detection agent directed against the toxigenic endophyte or a toxin produced by the toxigenic endophyte, wherein specific binding of the detection agent in the sample is indicative of colonization.

6. The method of claim 5, wherein the colonization comprises a toxigenic endophyte hosted by the seedling in an amount adequate to reduce growth rate, pest development and/or reduce pest weight gain, of spruce budworm larvae in a spruce budworm larvae assay compared to growth of spruce budworm in a non-inoculated conifer seedling.

7. The method of claim 1, wherein the toxigenic endophyte produces at least one toxin compound selected from the group consisting of rugulosin, vermiculin, 5-methoxy-carbonylmellein, 5-formylmellein, 5-methylmellin, 3-butyl-4-methylfuran-2(5H)-one butyrolactone tyrosol, mellein, 8-hydroxy-6-methoxy-3-proply-3,4-dihydroisocoumarin, or 3-methyl-5,7-dimethoxyphthalide, variants and plant modified forms thereof.

8. The method of claim 7, wherein the toxin compound is present in a conifer needle in an amount of at least 0.15 microgram per dram of needle or at least 10 micromolar.

9. The method of claim 1, wherein the conifer seedling is selected from a spruce seedling, a white spruce seedling, a red spruce seeding, a fir seedling and a pine seedling.

10. The method of claim 9, wherein the conifer seedling is susceptible when the seedling is at least 1 cm high and less than 10 cm high or 2 weeks to 16 weeks post germination.

11. The method of claim 9, wherein the white spruce seedling is inoculated with a white spruce toxigenic endophyte selected from the group of toxigenic endophytes deposit with Centraalbureau voor Schimmelcuitures (CBS) international depository agency in the Netherlands, having the accession numbers consisting of CBS 120377, CBS 120378, CBS 120381, CBS 120379, CBS 120380.

12. The method of claim 11, wherein the white spruce seedling is inoculated with a white spruce toxigenic endophyte deposited with Centraalbureau voor Schimmelcultures (CBS) international depository agency in the Netherlands, having the accession number CBS 120377.

13. The method of claim 11, wherein the white spruce seedling is inoculated with a white spruce toxigenic endophyte deposited with Centraalbureau voor Schimmelcultures (CBS) international depository agency in the Netherlands, having the accession number CBS 120378.

14. The method of claim 11, wherein the white spruce seedling is inoculated with a white spruce toxigenic endophyte deposited with Centraalbureau voor Schimmelcuitures (CBS) international depository agency in the Netherlands, having the accession number CBS 120381.

15. The method of claim 11, wherein the white spruce seedling is inoculated with a white spruce toxigenic endophyte deposited with Centraalbureau voor Schimmelcultures (CBS) international depository agency in the Netherlands, having the accession number CBS 120379.

16. The method of claim 11, wherein the white spruce seedling is inoculated with a white spruce toxigenic endophyte deposited with Centraalbureau voor Schimmelcultures (CBS) international depository agency in the Netherlands, having the accession number CRS 120380.

17. The method of claim 9, wherein the red spruce seedling is inoculated with a red spruce toxigenic endophyte selected from the group of toxigenic endophytes deposited with Centraalbureau voor Schimmelcultures (CBS) international depository agency in the Netherlands, having the accession numbers consisting of CBS 121946, CBS 121945, CBS 121944, CBS 121943, and CBS 12194.

18. The method of claim 17, wherein the red spruce seedling is inoculated with a red spruce toxigenic endophyte deposited with Centraalbureau voor Schimmelcultures (CBS) international depository agency in the Netherlands, having the accession number CBS 121946.

19. The method of claim 17, wherein the red spruce seedling is inoculated with a red spruce toxigenic endophyte deposited with Centraalbureau voor Schimmelcultures (CBS) international depository agency in the Netherlands, having the accession number CBS 121945.

20. The method of claim 17, wherein the red spruce seedling is inoculated with a red spruce toxigenic endophyte deposited with Centraalbureau voor Schimmelcultures (CBS) international depository agency in the Netherlands, having the accession number CBS 121944.

21. The method of claim 17, wherein the red spruce seedling is inoculated with a red spruce toxigenic endophyte deposited with Centraalbureau voor Schimmelcultures (CBS) international depository agency in the Netherlands, having the accession number CBS 121943.

22. The method of claim 17, wherein the red spruce seedling is inoculated with a red spruce toxigenic endophyte deposited with Centraalbureau voor Schimmelcultures (CBS) international depository agency in the Netherlands, having the accession number CBS 12194.

23. The method of claim 1, wherein the pest is selected from the group consisting of spruce budworm, hemlock looper, spruce budmoth, and disease causing fungal pathogen.

24. The method of claim 23, wherein the pest is a disease causing fungi is white pine blister rust.

25. The method of claim 1 wherein pest weight growth is inhibited by at least 5%, relative to a control.